Human papillomaviruses (HPVs) are common papilloma (wart)-causing viruses. They are responsible for inducing a variety of cutaneous and mucosal lesions and importantly are the cause of cervical and a subset of oropharyngeal cancers. Cervical cancer is the second-highest cancer-related cause of death among women worldwide. Although a vaccine has been approved by the FDA, cervical cancer remains a threat for the millions of women currently infected with genital HPVs. The majority of HPV-associated carcinomas harbor integrated virus, with increased expression of the viral oncogenes E6 and E7. The viral proteins interact with a number of cellular proteins, enhancing cellular proliferation through deregulation of cell cycle control mechanisms. A novel class of gene regulators called microRNAs (miRNAs) has recently been linked to many cancers and may play a role in progression to cancer. MiRNAs are 22 nt non-protein-coding RNAs that function as negative post-transcriptional gene regulators. MiRNAs hybridize to target messenger RNAs (mRNAs) and mediate translational repression or mRNA cleavage/destruction. MiRNA targets include genes involved in development, cell growth, and cell proliferation. Also, viruses involved in carcinogenesis have been found to encode miRNAs. The long term goals of this project are to gain a better understanding of the interaction between HPV-16, which is associated with cervical and oral cancers, and the cell during carcinogenesis and to identify diagnostic and therapeutic targets based on our miRNA studies. We hypothesize that HPV-16 affects the expression of cellular miRNAs, and we further hypothesize that HPV-16 encodes miRNAs which may target cellular and viral genes. The specific aims of this proposal are: (i) To determine how HPV-16 affects the expression of cellular miRNAs. Initial miRNA microarray studies showed differential expression of miRNAs between HPV-16 positive and HPV-16 negative cell lines. These data were validated by Northern blotting and qRT-PCR analyses. Transfection and siRNA studies showed that microRNA-218 was specifically reduced by HPV-16 E6 in cervical and oral carcinoma cells. Additional expression and protein analyses will be performed in order to characterize the role of HPV-16, particularly that of E6 and E7, in the regulation of miRNAs;(ii) To identify HPV-16-encoded miRNAs and their targets. Computational analysis with the RNA-folding software mFold suggests that HPV-16 may encode miRNAs. A custom HPV-16 microarray has been designed that encompasses the HPV-16 genome in both orientations. MiRNA fractions from HPV-16 positive and HPV-16 negative cell lines will be used as probes to identify viral miRNAs. We will then identify the cellular targets through computational and experimental methods.